Transforming growth factor (TGF)-[unreadable] is a critical regulator of events leading to chronic progressive renal disease such as extracellular matrix production, apoptosis and epithelial-mesenchymal transition (EMT). While considerable progress has been made in understanding signaling from the TGF-[unreadable] receptor (T[unreadable]R) through the Smad signaling molecules, the role of adaptor proteins such as Smad Anchor for Receptor Activation (SARA) in these signals is not well understood. Our preliminary data indicate that, in human mesangial cells (MC) and renal tubular epithelial cell lines (HKC and HK-2), SARA binds to Smad2 in a TGF-[unreadable]-dependent manner. In contrast, SARA is constitutively bound to T[unreadable]R and is associated with early endosomes. After TGF-[unreadable]1 treatment, Smad2 is phosphorylated and clathrin-mediated endocytosis (CME) facilitates propagation of the TGF-[unreadable] signal to the nucleus. Thus, SARA may play a central role in protein trafficking related to TGF-[unreadable] signaling, particularly through its actions in T[unreadable]R trafficking and promoting Smad2 activity. In addition to SARA, our preliminary data have detected other adaptor proteins in MC and HKC that are proposed to function in Smad signaling but are even less well understood than SARA. Prolonged TGF-[unreadable]1 treatment leads to an eventual decline in SARA expression, which corresponds temporally to the assumption of a myofibroblastoid phenotype in MC, HKC and HK-2. We propose the hypothesis that by acting as scaffolding proteins to assemble and localize the T[unreadable]R signaling complex, the adaptor proteins SARA, Dab2 and Axin may each impact the TGF-[unreadable] response, and the loss of SARA expression may alter the balance in T[unreadable]R interactions to modulate Smad signaling and enhance TGF-[unreadable]-stimulated fibrosis. To address this hypothesis we will pursue four specific aims: (1) Characterize the interactions of SARA, Dab2 and Axin with T[unreadable]R in TGF-[unreadable] signaling, their role in T[unreadable]R-complex trafficking, and their effect on Smad2- vs. Smad3-mediated responses to TGF-[unreadable]1. (2) Examine whether the loss of SARA is sufficient to induce alterations in signaling or trafficking related to fibrosis, and determine how these changes occur. (3) Define the mechanism by which TGF-[unreadable]1 decreases SARA expression and the role of PI- 3-kinase in this down-regulation. (4) Determine whether a decrease in SARA expression could play a role in established in vitro and in vivo models of renal fibrogenesis. These studies will identify novel events related to TGF-[unreadable] signaling and renal cell fibrogenic activity. SIGNIFICANCE: Although TGF-[unreadable] is an important mediator of renal fibrogenesis, its mechanisms of action are poorly understood. Our data suggest that the balance among several T[unreadable]R-interacting adaptor molecules plays an important role in regulating TGF-[unreadable] responses. Specifically, these studies may identify novel mechanisms whereby kidney cells transition to a fibrogenic phenotype. Understanding these mechanisms may indicate potential targets for interrupting renal scarring. PUBLIC HEALTH RELEVANCE Transforming growth factor (TGF)-[unreadable] is a protein that is found throughout the body and plays a central role in scarring of the kidney and other organs. Although we have made much progress in understanding how TGF-[unreadable] works, we have not learned how to regulate its actions in order to prevent or minimize scarring of the kidney. This project seeks to understand how one group of proteins in the cell, the TGF-[unreadable] receptor adaptor proteins, helps determine whether our cells are disposed to produce scar. In particular, we are interested in understanding how the expression of one protein, called SARA, is regulated and what role this regulation plays in scarring. Carrying out our specific aims will thus provide new basic information regarding how TGF-[unreadable] works and how the kidney is damaged in progressive kidney disease that could be useful in prevention and treatment.